Microbial electrode and microbial sensor

ABSTRACT

The microbial electrode is constructed having a substrate(3) made of an insulating material, an electric conductor(2) which is fixed on the substrate and a membrane containing microorganism cells(1) which is fixed on the conductor. The microbial sensor(20) is constructed having the microbial electrode above described and a conductor(11) to act as a counter electrode fixed on the insulating material of the surface of the substrate of the electrode which is opposite to the surface where the membrane containing microorganism cells is fixed.

TECHNICAL FIELD

The present invention relates to a microbial electrode and a microbialsensor, and more specifically to the microbial electrode and themicrobial sensor which enable measurement of the concentration of asample without resorting to oxygen electrodes.

BACKGROUND ART

The majority of microbial sensors which measure the concentration of amatter, measure the concentration of a matter in various solutions byutilizing a change of the microbial respiration. When microorganisms areput in a sample solution containing various matters and they metabolizethose matters, their respiration is activated intensely, which will leadto reduced oxygen concentration in the environment surrounding them.Such changes in oxygen concentration are measured by oxygen electrodes,and this is the method by which a microbial sensor can measure theconcentration of a substance.

For example, BOD (biochemical oxygen demand) is an important factor tobe considered when the quality of water in rivers and sewage from plantsmust be controlled, and has been taken internationally as an indicatorof the organic pollution of a water. The water pollutants derived fromorganic compounds are degraded through oxidation by aerobicmicroorganisms and consumed in the end, and in correspondence with thereduced concentration of those organic matters, dissolved oxygen is alsoconsumed. The measurement of how much oxygen is consumed represents thepollution of a given water. In other words, BOD represents theconcentration of organic compounds in terms of the oxygen consumed. Amethod for measuring BOD is regulated by the Japanese IndustrialStandard, JIS K 0102. However, this method requires an intricateoperation and it takes five days to measure BOD by this method, andhence it comes to be replaced the JIS K 0102 method by a method whichallows a quick, simple and on-line measurement of BOD (JIS K 3602⁻¹⁹⁹⁰).As a BOD sensor for this kind of method, a BOD sensor which depends onthe use of a combination of microbial film and oxygen electrodes(Japanese Laid-Open Patent Application No. 54-47699, etc.), has beenprovided and has been utilized for the measurement of BOD in industrialwaste water or the like.

Besides BOD sensors described above, the sensor utilizing microbialactivity includes a developed ethanol sensor which combines a membraneupon which are immobilized microorganisms such as Trichosporon brassicaewhich selectively consume ethanol and thus breathe vigorously in thepresence of ethanol, and oxygen electrodes which measure the reductionin oxygen concentration in a solution which occurs as a result ofactivated respiration.

However, in the sensor described above which determines theconcentration of matters in a solution by measuring the reduction indissolved oxygen with oxygen electrodes, there is a problem that it isdifficult to determine exactly the concentration of matters of asolution where dissolved oxygen remains at a low level. In addition, asthe oxygen electrode must contain an electrolyte and the like within itsstructure, it is forced to have a certain size. Accordingly, theconventional microbial sensors which are often installed into afermenter or the like to determine the concentration of various mattersand BOD sensors described above are all large or medium in size andexpensive.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is made in view of the above, and it is thereforean object of the present invention to provide a microbial sensor whichallows measurement even in a solution poor in dissolved oxygen, enablesdirect measurement of the concentration of matters in a solution withoutresorting to oxygen electrodes, thereby being capable of down sizing,and a microbial sensor which can be provided with reduced cost asproduction, and is disposable and free from reduced precision inmeasurement due to deteriorated microorganisms after long use.

The inventor(s) of the present invention, to solve the above mentionedproblems, constructed a microbial electrode or a microbial sensor havingthe following structure.

That is, the present invention provides a microbial electrode whichcomprises: a substrate made of an insulating material; an electricconductor which is fixed on the substrate; and a membrane containingmicroorganism cells which is fixed on the conductor, or a microbialelectrode which comprises: a substrate made of an electric conductor,and a membrane containing microorganism cells which is fixed on thesubstrate, and which is totally covered with an insulating coat exceptat least a part of the membrane containing microorganism.

Further, the present invention provides a microbial sensor characterizedby comprising: the above mentioned microbial electrode and; a conductorto act as the counter electrode and fixed on the insulating material ofthe surface of the substrate of the electrode which is opposite to thesurface where the membrane containing microorganism cells is fixed. Thepresent invention further provides a microbial sensor characterized bycomprising: the above mentioned microbial electrode; a substrate made ofan insulating material arranged with facing to the membrane containingmicroorganism cells of the electrode; a conductor to act as the counterelectrode and fixed on the surface of the substrate facing to themembrane containing microorganism of the substrate; and a sample liquidholding member having absorption property inserted and fixed between theconductor and the membrane containing microorganism.

Still further, the present invention provides a microbial sensorcharacterized by comprising: a substrate made of an insulating material;two conductors fixed on one surface of the substrate so as not to haveany physical contact with each other; and a membrane containingmicroorganism which is fixed on one of the two conductors, or amicrobial sensor which comprises: a first substrate; a first conductorto act as an electrode and fixed on the surface of the first substrate;a second substrate with an opening and made of an insulating material,which, facing through the opening on the first conductor, covers thefirst conductor; a second conductor to act as the counter electrodefixed on the surface of the second substrate; a membrane containingmicroorganism which is fixed on the first conductor and inside of theopening of the second substrate; and a frame which having an openingforming a sample liquid accumulation portion and facing through theopening on the membrane containing microorganism and the secondconductor, covers the second conductor.

Furthermore, the present invention provides a sample liquid collectingunit characterized by comprising: a sample liquid sucking inlet; aliquid accumulation portion for storing sample liquid sucked through thesample liquid sucking inlet; and an aspiration pump for generating anaspirating power, which incorporates above mentioned microbial sensor inthe sample liquid accumulation portion.

The present invention will be described in detail below.

<1> Microbial electrode of the present invention

The microbial electrode of the present invention is comprised of asubstrate made of an insulating material, an electric conductor fixed onthe substrate and a membrane containing microorganism cells which isfixed on the conductor.

The microbial electrode of the present invention, when used incombination with a counter electrode or with a counter electrode and areference electrode, may be utilized as a microbial sensor to measurethe concentration of matters in various kinds of solutions.

When microorganisms are allowed to exist in a sample solution containingorganic compounds, the microorganisms metabolize those organic compoundsto acquire energy. During the metabolysis, transfer of electrons occursin the electron transport system of its respiratory chain. Also, somemicroorganisms metabolize inorganic matters to acquire energy, andduring the metabolysis transfer of electrons occurs, too.

At this time, the concentration of matters metabolized correlates to theamount of electrons that are transferred. Therefore, it is possible todetermine the concentration of matters around the microorganisms bymeasuring the amount of transferred electrons. Since it is difficult todirectly measure the transferred amount of electrons, according to thepresent invention, a microbial electrode having the above describedstructure is immersed in a sample solution in combination with a counterelectrode or with a counter electrode and a reference electrode, and acertain potential difference is applied between the microbial electrodeacting as the working electrode and the counter electrode or thereference electrode such that the electrons easily transfer to measurethe current flowing between both the electrodes so that theconcentration of matters in the sample solution can be determined. Thestructure of the microbial electrode of the present invention will bedescribed below.

The microorganism to be applied to the electrode of the presentinvention includes any kind of microorganisms as long as they allowelectrons to tranfer through the electron transport system while theymetabolize organic or inorganic matters, and should not be limited toany specific microorganisms. The microorganism may include a procaryoteand an eucaryote, but the procaryote is more preferred, as far as thesensitivity in the measurement of the concentration of matters in asolution is concerned, than the eucaryote because in cells of the latterthe electron transfer through the electron transport system of itsrespiratory chain is conducted in the mitochondria which makes itdifficult to readily detect the amount of electrons that transfer to theworking electrode (microbial electrode) as the electric current.

When the microbial electrode of the present invention is applied fordetermination of the concentration of matters in a solution, it ispreferable to choose appropriate microorganisms which can metabolize thematters the concentration of which is to be determined. Further, whenthe concentration of one matter out of various kinds of matterscontained in a solution is to be measured, it may be prepared amicrobial electrode with using a microorganism which can metabolize thematter whose concentration is to be determined. Thus, there areappropriate combinations between matters to be determined andmicroorganisms to metabolize them. For BOD determination, it may beavailable for the microorganism to allow electrons to transfer throughthe electron transport system while it metabolizes organic matters, andsuch microorganism may include procaryotes such as Escherichia coli,bacteria of belonging to the genus Bacillus, genus Acinetobacter, genusGluconobacter, and genus Pseudomonas, and actinomycetes and eucaryotessuch as yeast of belonging to the genus Trichosporon. Otherrecommendable combinations between matters to be determined andmicroorganisms to metabolize them are cited below.

Pseudomonas aerginosa and the like for polyethyleneglycol; Pseudomonascepacia, Pseudomonas fulva and the like for phthalate esters;Acinetobacter, Pseudomonas putida, Pseudomonas paucimobilis and the likefor polybiphenylchloride; Pseudomonas putida and the like for phenol;Clostridium cochlearium and the like for organomercury compounds;Methylomonas methylovara, Hansenula polymorpha and the like formethanol; Hansenula angusta, Candida mycoderma, Gluconobacterrubiginosus IFO3244 and the like for ethanol; Pseudomonas aerginosa andthe like for cadmium; Pseudomonas putida, Pseudomonas convexa and thelike for toluene; Pseudomonas ovalis and the like for m-xylene;Desulfovibrio deseulfuricans, Thiobacillus thiooxidans, Thiobacillusthioparus and the like for organosulfur; Pseudomonas alcaligenes and thelike for dibenzothiophene; Clostridium butyricum and the like for formicacid; Methylococus capsulatus, Methylosinus trichosporium and the likefor methane gas; Arthrobacter petroleophagus, Mycobacteriumpetroleophilum and the like for ethane gas; Lactobacillus arabinosas andthe like for nicotinic acid; Brevibacterium cacaveris and the like foraspartic acid; Gluconobacter suboxydans IFO3172 and the like forglucose; Pseudomonas fluorescens IFO14160 and the like for glycerol;Pseudomonas putida IFO14164 for L-glutamic acid; Pseudomonaspseudomallei ATCC15682 and the like for maltose; Bcillus subtilusIFO13719 and the like for acetic acid; Pseudomonas caryophylli IFO13591and the like for sucrose; and Pseudomonas putida IFO14164 and the likefor S. starch. They are cited as appropriate combinations betweenmatters to be determined and microorganisms to metabolize them just forillustration, and the present invention is not limited to thosecombinations.

Such microorganisms as described above are incorporated into themicrobial electrode of the present invention, and the microorganism, tobe used as an electrode, is contained in a membrane. This microorganismcontaining membrane, for making the microorganism cells present on ornear the surface of the conductor as the electrode fixed on thesubstrate made of an insulating material, and may take any form as longas it is thin. Microorganism cells, for example, may be included into agel matrix of an alginate gel membrane, a carrageenan gel membrane, anagarose gel membrane, a curdlan gel membrane, a chitosan gel membrane,or the like, or into a photo-setting resin membrane such as aphoto-crosslinkable polyvinyl alcohol membrane, or into athree-dimensional crosslinked structure of a polyacrylamide membrane orthe like.

Further, microorganism cells may be immobilized with a polymer membrane.Furthermore, as another modification, microorganisms may be immobilizedwith glutaraldehyde or the like in membranous form on the surface of thesubstrate which is an element of the microbial electrode so thatelectric connection can be readily made with the conductor as theelectrode. As a further modification, microorganisms can be immobilizedusing an appropriate combination of above methods according to givenpurposes. Microorganisms in the membrane are preferably alive.

The substrate to form an element of the microbial electrode of thepresent invention supports the conductor acting as an electrode and themembrane containing microorganism cells, and is insulating because itmust prevent electric current from flowing between the above conductorand the counter electrode when the assembly is used a microbial sensor.In other words, the substrate to form an element of the microbialelectrode of the present invention may be made of any materials and isnot limited to any specific ones, as long as it is sufficiently strongto firmly support the conductor and the membrane containingmicroorganism cells and has an insulating property in a solution. It maybe made of, for example, plastics such as polyester, glass, and paperwhose surface has been so treated as to prevent the invasion of a samplesolution. According to the present invention, the substrate ispreferably made of a porous material because such material prevents themembrane containing microorganism cells from being peeled off.

The conductor acting as an electrode to form another element of themicrobial electrode of the present invention is fixed on the substratein such a manner that it is electrically connected to the membranecontaining microorganism cells, and receives electrons which aregenerated in association with the metabolic activity of themicroorganisms exposed to matters to be determined in a sample solution.The conductor may be made of any material as long as it is stable,highly electroconductive, and substantially innocuous to themicroorganisms, and may include metals such as platinum, gold andsilver, and carbonaceous materials such as graphite and carbon. It maytake any form, but is preferably shaped like a bar, a cylinder or asheet. It is preferably so prepared as to make the contact area with themembrane containing microorganism cells as large as possible.

For example, the conductor may be made into a conductive layer and befixed on one surface of the insulating substrate, thereby being possiblefor the conductor to have as large a contact area with the membranecontaining microorganism cells as possible. One example of the microbialelectrode includes one where a metal layer is formed by deposition onone surface of the substrate, and the membrane containing microorganismcells is fixed on the metal layer.

Further, the conductor of the microbial electrode of the presentinvention is installed in such a manner that it may not come intocontact with a sample solution directly. This is to prevent electriccurrent from flowing between the conductor in question and the counterelectrode when the microbial electrode of the present invention isimmersed into the sample solution. If the structure of the microbialelectrode happens to allow part of its electric conductor to come intocontact with the sample solution, and electric current to leaktherethrough, that part must be electrically insulated with someinsulating material. For example, in the case where the electricconductor fixed on the substrate of the microbial electrode is exposedfrom the microorganism cell, to thereby have a part which comes intocontact with the sample solution, tinsulatedhat part must beelectrically insulated with insulating material such as an epoxy resin.

Here, it is possible to make the substrate and conductor of themicrobial electrode of the present invention from the same material asappropriate according to given purposes.

In other words, according to another aspect of the present invention,the microbial electrode has a structure comprising a substrate made of aelectroconductive material, and a membrane containing microorganismcells which is firmly fixed on the substrate, and has the whole coveredwith an insulating coat except at least a part of the membranecontaining microorganism cell.

This type of microbial electrode obviates the necessity of fixing aconductor to a substrate as in the above described microbial electrode,because it incorporates a substrate made of a conductive material. Thus,with this electrode, the substrate itself acts as an electrode. However,with this microbial electrode, it is necessary to electrically insulatethe surface on which the membrane containing microorganism cells is notfixed, that is, all the part of the substrate which may possibly comeinto contact with a sample solution directly, so that electric currentcan be effectively prevented from flowing between the conductor and thecounter electrode when the microbial electrode is immersed into thesample solution.

In the actual use, which one of the two aspects of microbial electrodesof the present invention should be selected is appropriately determinedin consideration of the kinds of the conductor or the like.

A microbial sensor incorporating above described microbial electrode ofthe present invention comprises, in addition to the microbial electrodeacting as the working electrode, a counter electrode, and further areference electrode as needed. The counter electrode may be made ofplatinum, silver, gold, carbon or the like. It sometimes happens that,when the microbial sensor is immersed into a measurement sample solutionand the potential difference is applied between the working electrode(microbial electrode) and the counter electrode, the concentration ofthe reaction materials on the surface of the electrode decreases, andthe concentration of the products increases as the reaction of theelectrode proceeds. This may lead to a shift of the potential of theelectrode difference from the set potential. Therefore, it is preferredto immerse a reference electrode such as an Ag/AgCl electrode or thelike into a sample solution, and to set the potential of the workingelectrode on the basis of the reference electrode (3-electrode method).

Furthermore, when a sample solution contains a plurality of matters tobe determined, it is desirable and possible to incorporate a pluralityof microbial electrodes each comprising a membrane containingmicroorganism corresponding to the individual matter to be determined,and the counter electrodes, and further the reference electrodes asneeded, into a microbial sensor which allows the synchronous measurementof a plurality of matters contained in the same sample solution.

<2> Microbial sensor and sample liquid collecting unit of the presentinvention

The microbial sensor of the present invention may be produced such that,as described above, a microbial electrode, a counter electrode and areference electrode as needed may be formed separately. However,according to the present invention, the microbial electrode and thecounter electrode are integrally formed so as to form the microbialsensor in a small size or to simplify the measurement.

To obtain the microbial sensor in which the microbial electrode and thecounter electrode is integrally formed as described above, the microbialsensor of the present invention has the following structure.

According to present invention, the microbial sensor is comprised of themicrobial electrode of the present invention described above, and aconductor to act as the counter electrode which is fixed on theinsulating material of the surface of the substrate of the electrodewhich is opposite to the surface on which the membrane containingmicroorganism cells is fixed.

Alternatively, the microbial sensor of the present invention iscomprised of the above mentioned microbial electrode, a substrate madeof an insulating material arranged with facing to the membranecontaining microorganism cells of the electrode, a conductor to act asthe counter electrode and fixed on the surface of the substrate facingto the microorganism containing membrane of the substrate, and a sampleliquid holding membre having absorption property inserted and fixedbetween the conductor and the membrane containing microorganism cells.Since the microbial sensor with the sample liquid holding member isintegrally formed of the microbial electrode and the counter electrode,and allows the holding member to hold sample liquid, the microbialsensor may be used, for example, for the measurement of theconcentration of a matter contained in a small volume of a sample liquidkept at the bottom of a beaker. Further, a sample liquid may becollected with this microbial sensor itself, for example, from a bigtank, and may be immediately subjected to measurement, thus isconvenient.

These microbial sensors are constructed so that the conductor of themicrobial electrode may not make a direct contact with a sample solutionto prevent, as discussed above, electric current from flowing directlybetween the microbial electrode and the counter electrode. Otherwise,when the conductor of the microbial electrode structurally includes apart which may directly contact with a sample solution, that part iselectrically insulated with an insulating coat made of an epoxy resin orthe like so that a leak current may not flow therethrough. Suchinsulating coating treatment may also be applied to the conductor actingas the counter electrode so that the counter electrode may have the samecontact area with a sample solution whenever it is immersed into thesolution.

Furthermore, another aspect of a microbial sensor in which a microbialelectrode of the present invention and a counter electrode areintegrally formed, includes a microbial sensor comprising a substratemade of an insulating material, two conductors fixed on one surface ofthe substrate so as not to have any physical contact with each other,and a membrane containing microorganism cells which is fixed on one oftwo conductors described above. Of these two conductors which are sofixed on the substrate of the microbial sensor as not to physicallycontact with each other, one acts as the counter electrode as it is,while the other conductor, on which the membrane containingmicroorganism cells being fixed as in the above mentioned microbialelectrode of the present invention, acts as the microbial electrode(working electrode). In other words, this microbial sensor of thepresent invention includes the microbial electrode and the counterelectrode on the same surface of the same substrate.

Further, in this type of microbial sensor of the present invention, aspacer having a sample liquid pouring inlet and a cover are laminated onthe surface of the substrate on which a membrane containingmicroorganism cells is formed, to thereby form a sample liquid holdingportion which is surrounded with the surface of the substrate on which aconductor and a membrane containing microorganism cells are formed, theinternal wall of the spacer, and the lower edge of the cover.Furthermore, in the microbial sensor of the present invention, a sampleliquid holding member having absorption property is provided in thesample liquid holding portion, to thereby provide the microbial sensorwhich enables a sample liquid to be spontaneously injected into thesensor to measure the concentration of the matter therein. In this case,furthermore, it is preferred to install a filter onto the sample liquidpouring inlet so that the measurement is not interfered by floatingmatters or the like in the sample liquid which is poured through theinlet and adhere onto the electrodes. Further, the cover preferably hasan air vent which faces on a part of the sample liquid holding portionso that the sample liquid may smoothly enter thereto.

Further, when this microbial sensor is applied for the simultaneousmeasurement of the concentration of various matters to be measured andcontained in a sample solution, the number of conductors to be formed soas not to contact with each other on the substrate of the microbialsensor should be the number of matters to be measured plus one (onerepresents the number of the conductor to act as a counter electrode),and to each conductor to act as an working electrode (excluding thecounter electrode) is fixed a membrane containing a microorganismappropriate for the measurement of a matter to be measured.

In this case, according to the microbial sensor of the presentinvention, the conductor to act as the microbial electrode is mounted sothat it may not have a direct contact with a sample solution, when themicrobial sensor is immersed into the sample solution, in order toprevent electric current from directly flowing between the conductor toact as the microbial electrode and the conductor to act as the counterelectrode. Further, when the conductor of the microbial electrodestructurally includes a part which may directly contact with a samplesolution, that part is electrically insulated with an insulating coatmade of an epoxy resin or the like. Similar treatment such as theinsulating coating may also be applied to the conductor to act as thecounter electrode so that the counter electrode may have the samecontact area with a sample solution whenever it is immersed into thesolution.

Furthermore, on the surface of the substrate of the above mentionedmicrobial sensor on which a conductor (counter electrode) and amicrobial electrode (working electrode) are formed, a referenceelectrode is formed so as not to contact with the working electrode anda counter electrode with each other, thereby being capable of providinga microbial sensor incorporating three electrodes (working electrode,counter electrode and reference electrode) in one unit.

Such conductors to be used for the respective microbial sensors of thepresent invention may be made of the same materials as that of themicrobial electrodes of the present invention. Also the insulatingsubstrate onto which the conductors are fixed, may be made of the samematerial as that of the microbial electrode of the present invention.According to one aspect of a microbial sensor of the present invention,a sample liquid holding member having absorption property is insertedfor fixing between a conductor to act as a counter electrode and amembrane containing microorganism cells. According to another aspect ofa microbial sensor of the present invention, a sample liquid holdingportion in which a sample liquid holding member having absorptionproperty is provided, is installed as a part of the microbial sensor inthe sensor. The sample liquid holding member may be made of anymaterials, as long as the material can absorb a give amount of sampleliquid and hold it, and is not limited to any specific materials. Thematerial includes, for example, sponge, nylon mesh and the like.Further, the spacer and the cover which are used in a microbial sensorhaving a sample liquid holding portion within its structure as a part ofthe microbial sensor may be made of the same materials as are used forthe insulating substrate of the microbial electrode of the presentinvention.

When such microbial sensor is used for the measurement of theconcentration of matters contained in a sample solution, the microbialsensor is immersed into the measurement sample solution, a certainpotential difference is applied between the working electrode (microbialelectrode) and the counter electrode so that the electrons may transfereasily between them, and an electric current flowing between both theelectrodes is measured, thereby being capable of measuring theconcentration of matters in the sample solution. Also in this case, asdescribed above, it is possible to immerse a reference electrode such asan Ag/AgCl electrode or the like into the sample solution together withthe microbial sensor, and to set the potential of the working electrodeon the basis of the reference electrode.

Furthermore, according to the present invention, another aspect of amicrobial sensor in which a microbial electrode and a counter electrodeare integrally formed into one unit, is comprised of a first substrate,a first conductor to act as an electrode and fixed on the surface of thefirst substrate, a second substrate with an opening and made of aninsulating material, which, facing through the opening on the firstconductor, covers the first conductor, a second conductor to act as thecounter electrode fixed on the surface of the second substrate, amembrane containing microorganism cells which is fixed on the firstconductor and inside of the opening of the second substrate, and a framewhich having an opening forming a sample liquid accumulation portion andfacing through the opening on the microorganism containing membrane andthe second conductor, covers the second conductor.

The membrane containing microorganism cells which is incorporated intothe microbial sensor of the present invention may be made of the samematerials as are used for the membrane containing microorganism cells ofthe microbial electrode of the present invention. Further, the firstconductor to act as an electrode and the second conductor to act as acounter electrode of the microbial sensor of the present invention maybe made of the same materials as those for the conductors of themicrobial electrode of the present invention. Furthermore, the firstsubstrate, the second substrate, and the frame of the microbial sensormay be made of the same insulating materials as are used for thesubstrate of the microbial electrode of the present invention. However,the first substrate is not always made of an insulating material.

When this microbial sensor is used for the measurement of theconcentration of matters in a sample solution, the sample solution isallowed to enter into the opening constituting a sample liquidaccumulation portion by that amount which allows the sample solution tosufficiently come into contact with the membrane containingmicroorganism cells and the second conductor, and which, on the otherhand, to prevent it from spilling over the frame disposed on the secondconductor from the sample liquid accumulation portion. The samplesolution osmoses into the membrane containing microorganism cells whichis fixed on the first conductor which is inside of the opening of thesecond substrate, and then the microorganism metabolizes the matter inthe sample solution within the membrane. This metabolism is associatedwith tansfer of the electrons occuring in the respiratory system of themicroorganism, but in this sensor, instead of measuring directly thiselectrons transfer, a certain potential difference is applied betweenthe first conductor to act as an electrode on which the membranecontaining microorganism cells is fixed and the second conductor to actas a counter electrode and directly contact with a sample solution, sothat the electrons easily transfer, and then an electric current flowingbetween both the electrodes is measured, thereby being capable ofdetermining the concentration of the matter in the sample solution.

It is also possible to add a detachable or a fixed cover to themicrobial sensor to prevent a sample liquid injected into the sampleliquid accumulation portion from being dried during a measurement of theconcentration of the matter. When the cover is fixed, it is necessary toadd a passage to an appropriate place, for example, at the frame, bywhich the sample liquid can enter from outside of the microbial sensorinto the sample liquid accumulation portion. Furthermore, in this case,the cover preferably has an air vent which faces on a part of the sampleliquid accumulation portion, as same as the cover of the above describedmicrobial sensor acting through capillary action. This is to facilitatesmooth inflow of the sample liquid into the sample liquid accumulationportion.

According to the present invention, furthermore, taking advantages uponmeasurement of the concentration of matters in various sample liquidsinto consideration, there is provided a sample liquid collecting unitincorporating therein the respective microbial sensors described above.

In other words, the sample liquid collecting unit of the presentinvention is characterized by comprising a sample liquid sucking inlet,a sample liquid accumulation portion for storing sample liquid suckedthrough the sample liquid sucking inlet, and an aspiration pump portionfor generating aspirating power, and incorporating a microbial sensor inthe sample liquid accumulation portion.

Examples of this kind of sample liquid collecting unit may include aunit that comprises an apparatus having a sample liquid accumulationportion which can precisely suck a constant volume of sample liquid,such as a piston cylinder-type one like a syringe or a pipette-type one,and a microbial sensor incorporated therein.

According to the microbial sensor of the present invention, a certainpotential difference is applied between an electrode on which themembrane containing microorganism cells is fixed and the counterelectrode, and then an electric current flowing between both theelectrodes is measured. For application of the potential difference andthe measurement of the current, it is preferred to use a constantvoltage generator, a potentiostat, or the like. Further, small-sizedmeasuring instruments appropriate for the present purpose including thepotentiostat or the like are commercially available. Thus, it ispossible for the above mentioned sample liquid collecting unit of thepresent invention, or the like, to connect the microbial sensor with asmall-sized measuring instrument such as a small potentiostat, and toincorporate it into the sample liquid collecting unit.

Further, an electron transport mediator (refer to as simply "mediator"hereinafter) is preferably added to a sample liquid, because it allows ahigher sensitivity measurement. Otherwise, the mediator may beintroduced into the interface with the sample liquid, such as theinterior or surface of the membrane containing microorganism, a portionbetween the membrane containing microorganism and the electrode, or thelike. It should be noted that when a mediator is used while being fixedon a microbial electrode, the mediator will elute into the sample liquidduring measurement of the concentration of sample liquid. Therefore,with such microbial sensor, it is preferred to make the microbial sensorused one time disposable, or to replenish the sensor with fresh mediatorafter use for the next use.

The mediator facilitates, the electrons generating from the metabolismof the various matters by the microorganism to transfer to theelectrode. The mediator may be composed of any materials, and is notlimited to any specific materials as long as it facilitates the transferof electrons from the microorganism to the electrodes. Examples of themediator include pigments such as 1-methoxy-5-methylphenaziniummethylsulfonate (1-M-PMS), 2,6-dichloroindophenol(DCIP),9-dimethylaminobenzo-a-phenazoxonium chloride, methylene blue,indigotrisulfonic acid, phenosafranin, thionine, new methylene blue,2,6-dichlorophenol, indophenol, azule B, N, N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride, resorufine,safranine, sodium anthraquinone β-sulfonate, and indigo carmine;biological oxidation/reduction materials such as riboflavin, L-ascorbicacid, flavin adenine dinucleotide, flavin mononucleotide, nicotineadenine dinucleotide, lumichrome, ubiquinone, hydroquinone,2,6-dichlorobenzoquinone, 2-methylbenzoquinone,2,5-dihydroxybenzoquinone, 2-hydroxy-1,4-naphtoquinone, glutathione,peroxidase, cytochrom C and ferredoxin or their derivatives; and otherssuch as Fe-EDTA, Mn-EDTA, Zn-EDTA, mesosulfate,2,3,5,6-tetramethyl-p-phenylenediamine, potassium ferricyanide. Themediator preferably has a concentration of about 40 nM-100 mM, morepreferably, about 10 μM-50 mM.

Of those compounds, 1-M-PMS, DCIP, potassium ferricyanide and9-dimethylaminobenzo-α-phenazoxonium chloride are preferred.

Here, the method of measuring concentrations of matters contained invarious sample solutions using the above-described respective microbialsensors will be briefly outlined below, taking as an example themeasurement of BOD.

The measurement of BOD consists of drawing a standard curve using astandard sample, and then finding the BOD of a sample liquid from thevalue of electric current obtained with the use of the sample liquid. Inother words, by using a buffer solution free of organic compounds, anelectric current flowing between the working electrode, and the counterelectrode or the reference electrode is measured, and the value ofelectric current is measured using different concentrations of standardsample to draw a standard curve. Subsequently, the measurement ofelectric current is conducted using a measuring sample or a measuringsample liquid diluted with the buffer solution in the same manner as inthe above. These measurement values of electric currents are comparedwith the values of electric currents obtained using the standard sample,to thereby measure the BOD. Further, when the microbial sensor which hasa mediator and a buffer solution stabilized on the membrane containingmicroorganism is used for the measurement, the sensor is directlyimmersed into the sample liquid, an electric current flowing between theworking electrode (microbial electrode) and the counter electrode or thereference electrode is measured. The measurement values are comparedwith the values of electric currents obtained using a standard sample,to thereby measure the BOD.

The current flowing through the sensor depends on different parametersincluding the kind of microorganism, the contact area between theelectrode and the microorganism membrane, the kind and the concentrationof the mediator, the potential difference applied between the workingelectrode and the counter electrode, the concentration of BOD and thelike. Therefore, these parameters may be determined as appropriateaccording to a given purpose after a preliminary experiment has beenmade.

When the BOD sensor of the present invention is immersed into a solutioncontaining the organic compounds, those organic compounds aremetabolized by the microorganism in the microorganism membrane of thesensor. Consequently, electrons transfer via the electron transportsystem. When the potential difference is applied between the workingelectrode and the counter electrode, electron transfers from themicroorganism membrane to the working electrode. As a result, thecurrent obtained is different from that given when electrons are notgenerated. The concentration of the organic compounds, that is, BOD canbe measured by measuring the electric current.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing one embodiment of a microbial electrodeaccording to the present invention; FIG. 1(a) is a perspective view andFIG. 1(b) is a sectional view thereof.

FIG. 2 is a diagram showing one embodiment of a microbial sensoraccording to the present invention; FIG. 2(a) is a perspective view andFIG. 2(b) is a sectional view thereof.

FIG. 3 is a diagram showing another embodiment of the microbial sensoraccording to the present invention; FIG. 3(a) is a perspective view andFIG. 3(b) is a sectional view thereof.

FIG. 4 is a sectional view showing another embodiment of the microbialelectrode according to the present invention.

FIG. 5 is a exploded perspective view of different embodiment of themicrobial sensor according to the present invention.

FIG. 6 is a diagram showing further different embodiment of themicrobial sensor according to the present invention; FIG. 6(a) is aassembling perspective view and FIG. 6(b) is a sectional view thereof.

FIG. 7 is a partially sectioned side view showing one embodiment of asample liquid collecting unit according to the present invention.

FIG. 8 is a front view showing a example of the BOD-measuring systemincorporating the microbial electrode according to the presentinvention.

FIG. 9 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withPVA-SbQ on a gold-deposited Omnimembrane substrate.

FIG. 10 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withPVA-SbQ on a gold-deposited polyester sheet substrate.

FIG. 11 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withPVA-SbQ on an Omnimembrane substrate having a carbon electrode.

FIG. 12 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withglutaraldehyde on a gold-deposited Omnimembrane substrate.

FIG. 13 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withPVA-SbQ and glutaraldehyde on a gold-deposited Omnimembrane substrate.

FIG. 14 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withagarose on a gold-deposited Omnimembrane substrate.

FIG. 15 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withagarose on a gold-deposited polyester sheet substrate.

FIG. 16 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withagarose having a low melting-point on a gold-deposited Omnimembranesubstrate.

FIG. 17 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withpolyacrylamide on a gold-deposited Omnimembrane substrate.

FIG. 18 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withcurdlan on a gold-deposited Omnimembrane substrate.

FIG. 19 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withcurdlan on a gold-deposited polyester sheet substrate.

FIG. 20 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withcurdlan and glutaraldehyde on a gold-deposited Omnimembrane substrate.

FIG. 21 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withcurdlan and glutaraldehyde on a gold-deposited polyester sheetsubstrate.

FIG. 22 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withcarrageenan on a gold-deposited Omnimembrane substrate.

FIG. 23 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withchitosan on a gold-deposited Omnimembrane substrate.

FIG. 24 is a graph showing results of BOD measurements using themicrobial electrode in which microorganism cells are immobilized withENT on a gold-deposited Omnimembrane substrate.

FIG. 25 is a graph showing a comparison of results of the BODmeasurements obtained by 2-electrodes method (the microbial electrodeand the counter electrode) and 3-electrodes method (the microbialelectrode, the counter electrode and the reference electrode) appliedfor the microbial electrode in which microorganism cells are immobilizedwith PVA-SbQ on a gold-deposited Omnimembrane substrate.

FIG. 26 is a graph showing results of BOD measurements using themicrobial electrode which incorporates a mediator with carboxymethylcellulose.

FIG. 27 is a graph showing results of BOD measurements using themicrobial electrode which incorporates a mediator withpolyvinylpyrrolidone.

FIG. 28 is a graph showing results of glucose concentration measurementsusing a microbial electrode according to the present invention.

FIG. 29 is a graph showing results of glycerol concentrationmeasurements using a microbial electrode according to the presentinvention.

FIG. 30 is a graph showing results of ethanol concentration measurementsusing a microbial electrode according to the present invention.

FIG. 31 is a graph showing results of L-glutamic acid concentrationmeasurements using a microbial electrode according to the presentinvention.

FIG. 32 is a graph showing results of maltose concentration measurementsusing a microbial electrode according to the present invention.

FIG. 33 is a graph showing results of acetic acid concentrationmeasurements using a microbial electrode according to the presentinvention.

FIG. 34 is a graph showing results of sucrose concentration measurementsusing a microbial electrode according to the present invention.

FIG. 35 is a graph showing results of S. starch concentrationmeasurements using a microbial electrode according to the presentinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will be described specifically hereinafter.

EXAMPLE 1 Microbial Electrode and Microbial Sensor for Measurement ofBOD

Firstly, one example of a microbial electrode of the present inventionwhich constitutes a BOD sensor and a BOD sensor incorporating thismicrobial electrode will be described on basis of FIGS. 1 to 3.

<Preparation of a microorganism suspension>

The Pseudomonas fluorescens IFO14160 strain was inoculated in 80 ml of aculture medium (pH 7.0) containing 0.3% K₂ HPO₄, 0.1% KH₂ PO₄, 0.03%MgSO₄.7H₂ O, 0.5% ammonium sulfate, 0.001% L-glutamic acid and 0.1%yeast extract, and cultivated at 30° C. for 48 hours with shaking. Theculture was centrifuged to separate the cells from the medium. The cellswere washed with a buffer solution (0.05 M phosphate, pH 7.0), andsuspended in the same buffer solution.

<Preparation of a microbial electrode>

On the central portion of one surface of a substrate of Omnimembrane 3(Millipore, JGWP-14225) was evenly deposited gold 2 constituting anelectrode. On this gold-deposited surface of Omnimembrane was coatedwith evenly a liquid which was produced after an appropriate amount ofPVA-SbQ solution was added to the above cell suspension, so that when amicrobial electrode was inserted into a sample liquid, the sample liquidmight not come into contact with the gold electrode, and the whole wasincubated in a dark place at 37° C. for three hours. Subsequently, afluorescent lamp was irradiated to allow PVA-SbQ to undergocross-linking reaction, to thereby form a membrane containingmicroorganism cells 1. If there were parts where the gold electrodemight come into contact with the sample liquid, they were insulated withan epoxy resin 4. Thus, a microbial electrode 10 is prepared (FIG. 1).

<Preparation of a microbial sensor>

The following two kinds of microbial sensors were prepared byincorporating the above-described microbial electrode 10.

The first microbial sensor was first prepared after gold 11 to act asthe counter electrode had been deposited evenly on the whole surface ofabove-described substrate of Omnimembrane 3 which was opposite to thesurface where the membrane containing microorganism cells was fixed, andpart of the gold-deposited surface was insulated with an epoxy resin 4in order to adjust the contact area of the counter electrode with asample liquid (FIG. 2).

The another type of microbial sensor will be described on basis of FIG.3. The conductor to act as the counter electrode was made of gold 11which was evenly deposited on one surface of a polyester film 12 to actas a substrate, and part of the gold-deposited surface was insulatedwith an epoxy resin 4 in order to adjust the contact area of the counterelectrode with a sample liquid. Furthermore, a sponge 14 to act as asample liquid holding member was inserted and fixed with an epoxy resinbetween the gold-deposited surface of the polyester film and themembrane containing microorganism cells of the microbial electrode 10obtained above.

EXAMPLE 2 Another Embodiment of a Microbial Electrode

Another embodiment of a microbial electrode of the present inventionwill be described below on basis of FIG. 4.

A membrane containing microorganism 1 was allowed to form on one surfaceof a carbon film 3 to act as a substrate (this also acts as a conductor2) in the same manner as in Example 1. The surface of the substratewhich comes into contact with a sample liquid was insulated with anepoxy resin 4 except the portion on which the membrane containingmicroorganism was formed. Thus, a microbial electrode 10 was prepared.

EXAMPLE 3 Different Embodiment of a Microbial Sensor

Different embodiment of a microbial sensor of the present invention willbe described below on basis of FIG. 5.

Gold was deposited on two spots so as not to contact with each other onone surface of a substrate 3 made of polyester. On one of thesegold-deposited portion, a membrane containing microorganism 1 was formedin the same manner as in Example 1, and the other was left as it was toact as a counter electrode 11. Further, within the gold-depositedportion 2 where the membrane containing microorganism was formed, anyparts of it which might come into contact with a sample liquid wasinsulated with an epoxy resin 4. Furthermore, part of the gold-depositedportion to act as the counter electrode was insulated with an epoxyresin 4 in order to adjust the contact area of the counter electrode 11with a sample liquid. The resulting product may be used as a microbialsensor when it is immersed into a sample liquid. The microbial sensorwas further modified as follows so that it could automatically absorbthe sample liquid through capillary action.

On the surface of the polyester substrate on which the membranecontaining microorganism 1 and the counter electrode 11 were formed, apolyester spacer 13 having a sample liquid pouring inlet 21 with afilter was laminated to form a sample liquid holding portion, and anylon mesh to act as a sample liquid holding member 14 was inserted intothe sample liquid holding portion. Furthermore, a polyester cover 15having an air vent 16 facing on a part of the sample liquid holdingportion was laminated over the spacer 13 and the nylon mesh 14, thus amicrobial sensor 20 utilizing a capillary action was prepared.

EXAMPLE 4 Further Different Embodiment of a Microbial Sensor

Further different embodiment of a microbial sensor will be describedbelow on basis of FIG. 6.

On a surface of a first substrate 17 made of polyester was depositedwith gold 2 to act as an electrode. On the gold-deposited surface of thefirst substrate 17, a second polyester substrate 18 having a roundopening, which facing through the opening on a part of thegold-deposited surface was laminated. On a surface of the secondsubstrate, a gold-deposited portion 11 to act as the counter electrodewas formed in advance.

A membrane containing microorganism 1 was fixed on the gold-depositedsurface on the first substrate and inside of the opening of the secondsubstrate in the same manner as in Example 1. Furthermore, a polyesterframe 19 having an opening to act as a sample liquid accumulationportion and a sample liquid injecting passage 22 for connecting outsideof the microbial sensor to the sample liquid accumulation portion 23,and facing through the opening on the membrane containing microorganismcells 1 and a part of gold-deposited surface of the second substrate 18,was laminated thereupon. Still further, a polyester cover 15 having anair vent facing on a part of the sample liquid accumulation portion 23was further laminated on the frame, to thereby prepare a microbialsensor 20.

EXAMPLE 5 Sample Liquid Collecting Unit

Into the body of a commercially available pipette (Pipettman, Gilson), aconstant voltage generator was installed and the microbial sensor 20obtained in Example 1 was connected and fixed thereto. A tip wasattached at the tip of the microbial sensor 20, and on the side of thebody, a current value display portion 25 for displaying the currentmeasured by the constant voltage generator was further installed toprovide a sample liquid collecting unit 40 (FIG. 7).

EXAMPLE 6 Example of BOD Measurement

By using the microbial electrode 10 as prepared in Example 1, BODmeasurement was carried out with the following measurement system (FIG.8). The above-described microbial electrode and a counter electrode(gold electrode) 11 were fixed to clips attached to a potentiostat 30,and the electrodes were immersed into a sample liquid tank 31 filledwith a buffer solution to thereby prepare a BOD sensor. The gold counterelectrode having its part insulated with an epoxy resin 4 to adjust itscontact area with a sample liquid was used. A certain voltage wasapplied between the microbial electrode 10 and the counter electrode 11,and the flowing current was measured. Next, the standard BOD liquid (amixed solution containing glucose and L-glutamic acid both at 150 mg/L(BOD at 220 mg/L), to be referred to as simply "BOD Standard liquid")was added together with a mediator (20 mM potassium ferricyanide (finalconcentration)), and the value of electric current was measured in thesame manner as in the above. During the BOD measurement, the sampleliquid was stirred with a magnetic stirrer 32. The measurements were fedto a computer 34. The results are shown in FIG. 9.

EXAMPLE 7 Various Immobilizing Methods and BOD Measurement

Further, the cell suspension of Pseudomonas fluorescens IFO14160 whichwas cultivated in the same manner as in Example 1 was immobilized onconductors fixed on various substrates, using various carriers asdescribed below, to prepare different microbial electrodes. The BODsensors comprising those microbial electrodes and counter electrodeswere installed into the same measurement system with that in Example 6,and applied to measurement of BOD.

(1) BOD sensor incorporating cells immobilized with PVA-SbQ

A microbial electrode was prepared in the same manner as in Example 1except that a polyester sheet was employed instead of the Omnimembraneas a material of the substrate, and applied for the measurement of BOD.For the measurement of BOD, 0.01 M phosphate buffer solution (pH 7.0) asa buffer solution and 80 nM 1-M-PMS as a mediator were used. The resultsare shown in FIG. 10.

Further, another microbial electrode was prepared in the same manner asin Example 1 except that a carbon electrode was used instead of the goldelectrode, and applied for the measurement of BOD. For the measurementof BOD, 0.01M phosphate buffer solution (pH 7.0) as a buffer solutionand 10 mM potassium ferricyanide as a mediator were used. The resultsare shown in FIG. 11.

(2) Glutaraldehyde

A gold-deposited Omnimembrane to act as a substrate was the same as inExample 1. On this gold-deposited surface of Omnimembrane, a solutionwhich was obtained after an appropriate amount of bovine serum albumin(BSA) was dissolved in the cell suspension obtained in Example 1 wascoated evenly, in such a manner that, when the electrode was immersedinto a sample liquid, the gold electrode might not come into directcontact with the sample liquid. The thus-obtained electrode was exposedto the vapor of a glutaraldehyde aqueous solution having a concentrationof about 25% of for 20 minutes to form a microorganism containingmembrane, and then rinsed three times with the phosphate buffer solutionto prepare a microbial electrode.

This microbial electrode was applied for the measurement of BOD. For themeasurement of BOD, 0.05 M phosphate buffer solution (pH 7.0) as abuffer solution and 20 mM potassium ferricyanide as a mediator wereused. The results are shown in FIG. 12.

(3) PVA-SbQ+glutaraldehyde

The microbial electrode incorporating the gold-deposited polyester filmsubstrate as obtained in above mentioned (1) was exposed to the vapor ofa glutaraldehyde aqueous solution having a concentration of about 25% offor 20 minutes, and rinsed three times with the phosphate buffersolution to prepare a microbial electrode.

This electrode was applied for the measurement of BOD. For themeasurement of BOD, 0.01 M phosphate buffer solution (pH 7.0) as abuffer solution and 80 nM 1-M-PMS as a mediator were used. The resultsare shown in FIG. 13.

(4) Agarose

Agarose was heat-dissolved to give an appropriate concentration (forexample 2%), and was maintained at about 60° C., and the cell suspensionobtained in Example 1 was added thereto and mixed. The resulting cellsuspension in agarose solution was evenly coated onto two kinds ofgold-deposited surfaces (gold-deposited polyester film substrate andgold-deposited Omnimembrane substrate) in such a manner that, when theywere immersed into a sample liquid, the gold electrodes might not comeinto direct contact with the sample liquid, and they were cooled toprepare microbial electrodes.

These two kinds of microbial electrodes were used for the measurement ofBOD. For the BOD measurement with the electrode incorporating thegold-deposited Omnimembrane substrate, 0.05 M phosphate buffer solution(pH 7.0) as a buffer solution and 20 mM potassium ferricyanide as amediator were used. The results are shown in FIG. 14. For the BODmeasurement with the electrode incorporating the gold-depositedpolyester film substrate, 0.01 M phosphate buffer solution (pH 7.0) as abuffer solution and 80 nM 1-M-PMS as a mediator were used. The resultsare shown in FIG. 15.

(5) Agarose having a low melting point

Agarose having a low melting point was heat-dissolved to give anappropriate concentration (for example 2%), and was maintained at about40° C., and the cell suspension obtained in Example 1 was added theretoand mixed. The resulting cell suspension in the solution of agarosehaving a low melting point was evenly coated onto the gold-depositedsurface of the gold-deposited Omnimembrane substrate in such a mannerthat, when it was immersed into a sample liquid, the gold electrodemight not come into direct contact with the sample liquid. Then, it wascooled to prepare a microbial electrode.

The microbial electrode was used for the measurement of BOD. For the BODmeasurement, 0.05 M phosphate buffer solution (pH 7.0) as a buffersolution and 20 mM potassium ferricyanide as a mediator were used. Theresults are shown in FIG. 16.

(6) Polyacrylamide

Polyacrylamide was heat-dissolved to give an appropriate concentrationand the cell suspension obtained in Example 1 was added thereto andmixed. The resulting liquid was evenly coated onto the gold-depositedsurface of the gold-deposited Omnimembrane substrate in such a mannerthat, when it was immersed into a sample liquid, the gold electrodemight not come into direct contact with the sample liquid. Then, it wascooled to prepare a microbial electrode.

The microbial electrode was used for the measurement of BOD. For the BODmeasurement, 0.05 M phosphate buffer solution (pH 7.0) as a buffersolution and 20 mM potassium ferricyanide as a mediator were used. Theresults are shown in FIG. 17.

(7) Curdlan

To 2% curdlan solution, the cell suspension obtained in Example 1 wasadded and mixed, and the mixture liquid was heated at 50 to 60° C. forabout one minute to melt curdlan. The resulting liquid was evenly coatedonto two kinds of gold-deposited surfaces (gold-deposited polyester filmsubstrate and gold-deposited Omnimembrane substrate) in such a mannerthat, when they were immersed into a sample liquid, the gold electrodesmight not come into direct contact with the sample liquid. Then, theywere cooled and gelated, and dried at room temperature to preparemicrobial electrodes.

These two kinds of microbial electrodes were used for the measurement ofBOD. For the BOD measurement with the electrode incorporating thegold-deposited Omnimembrane substrate, 0.05 M phosphate buffer solution(pH 7.0) as a buffer solution and 200 μM 1-M-PMS as a mediator wereused. The results are shown in FIG. 18. Further, for the BOD measurementwith the electrode incorporating the gold-deposited polyester filmsubstrate, 0.05 M phosphate buffer solution (pH 7.0) as a buffersolution and 20 μM 1-M-PMS as a mediator were used. The results areshown in FIG. 19.

(8) Curdlan+glutaraldehyde

The two kinds of microbial electrodes obtained in (6) described abovewere immersed into 2% glutaraldehyde aqueous solution and washedthoroughly, and then they were dried at room temperature.

These two kinds of microbial electrodes were used for the measurement ofBOD. For the BOD measurement with the electrode incorporating thegold-deposited Omnimembrane substrate, 0.05 M phosphate buffer solution(pH 7.0) as a buffer solution and 200 μM 1-M-PMS as a mediator wereused. The results are shown in FIG. 20. Further, for the BOD measurementwith the electrode incorporating the gold-deposited polyester filmsubstrate, 0.05 M phosphate buffer solution (pH 7.0) as a buffersolution and 20 μM 1-M-PMS as a mediator were used. The results areshown in FIG. 21.

(9) Carrageenan

To 1.5% carrageenan solution, the cell suspension obtained in Example 1was added and mixed, and the mixture liquid was evenly coated onto thegold-deposited surface of the gold-deposited Omnimembrane substrate insuch a manner that, when it was immersed in a sample liquid, the goldelectrode might not come into direct contact with the sample liquid.Then, it was cooled and gelated. Thereafter, the product was immersedinto 2% potassium chloride solution for several hours, and dried at roomtemperature to prepare a microbial electrode.

The microbial electrode was used for the measurement of BOD. For the BODmeasurement, 0.05 M phosphate buffer solution (pH 7.0) as a buffersolution and 5 mM potassium ferricyanide as a mediator were used. Theresults are shown in FIG. 22.

(10) Chitosan

To a chitosan solution which was prepared after 1-8% chitosan wasdissolved into 1-2% acetic acid aqueous solution, the cell suspensionobtained in Example 1 was added and mixed, and the mixture liquid wasevenly coated onto the gold-deposited surface of the gold-depositedOmnimembrane substrate in such a manner that, when it was immersed intoa sample liquid, the gold electrode might not come into direct contactwith the sample liquid. Then, it was dried at room temperature toprepare a microbial electrode.

The microbial electrode was used for the measurement of BOD. For the BODmeasurement, 0.05 M phosphate buffer solution (pH 7.0) as a buffersolution and 20 mM potassium ferricyanide as a mediator were used. Theresults are shown in FIG. 23.

(11) Photo-setting resin

1 g of photo-setting resin (ENT-2000, Kansai Paint) and 0.01 g ofphoto-polymerization inducer were mixed, and 0.5 g of the cellsuspension obtained in Example 1 was added thereto and mixed. Themixture liquid was evenly spread onto the gold-deposited surface of thegold-deposited Omnimembrane substrate in such a manner that, when it wasimmersed into a sample liquid, the gold electrode might not come intodirect contact with the sample liquid. Then, it was covered with apolypropylene film so that it might not contact with air, and light wasirradiated for polymerization. Thereafter, the polypropylene film wasremoved to prepare a microbial electrode.

The microbial electrode was used for the measurement of BOD. For the BODmeasurement, 0.05 M phosphate buffer solution (pH 7.0) as a buffersolution and 20 mM potassium ferricyanide as a mediator were used. Theresults are shown in FIG. 24.

From above results it is obvious that the BOD sensor of the presentinvention may be applied for the measurement of BOD regardless of themethod of immobilizing the microorganism cells onto the metal electrode.

EXAMPLE 8 Comparison of 2-Electrodes Method and 3-Electrodes Method

A microbial electrode was prepared by immobilizing Pseudomonasfluorescens IFO14160 onto a gold-deposited Omnimembrane substrate withPVA-SbQ in the same manner as in Example 1, and this microbial electrodewas combined with the counter electrode in 2-electrodes method, or withthe counter electrode and the reference electrode in 3-electrodes methodto measure BOD. Then they were used for the measurement of BOD.

In 2-electrodes method, the measurement was carried out in the samemanner as in Example 6 while in 3-electrodes method, the measurement wascarried out in the same manner as in Example 6 except that an Ag/AgClelectrode was used as a reference electrode. For these BOD measurements,0.05 M phosphate buffer solution (pH 7.0) as a buffer solution and 20 mMpotassium ferricyanide as a mediator were used. The results are shown inFIG. 25.

From this result it is obvious that it may be sufficiently available tomeasure the BOD whether with 2-electrodes method using only themicrobial electrode and the counter electrode, or with 3-electrodesmethod using the microbial electrode, the counter electrode and thereference electrode without the large difference of the results of themeasurement between both the methods.

EXAMPLE 9 Microbial Electrode Containing a Mediator

A microbial sensor was prepared which having a microbial electrode inwhich Pseudomonas fluorescens IFO14160 was immobilized on agold-deposited polyester substrate with PVA-SbQ in the same manner as inExample 4, except that, after the membrane containing microorganism wasformed, that membrane containing microorganism was allowed to contain amediator with the following method. Thus, a disposable, mediatorcontaining microbial sensor was obtained.

(1) Method for containing a mediator with carboxymethyl cellulose

Potassium ferricyanide as a mediator was dissolved in an appropriateamount of 1% concentration of carboxymethyl cellulose solution, spreadonto a membrane containing microorganism, and dried so that the mediatormight be incorporated into the membrane containing microorganism.

(2) Method for cntaining a mediator with polyvinylpyrrolidone

Potassium ferricyanide as a mediator was dissolved in an appropriateamount of about 10% concentration of polyvinylpyrrolidone solution,spread onto a membrane containing microorganism, and dried so that themediator might be incorporated into the membrane containingmicroorganism.

Mediator containing microbial sensors were prepared in the same manneras in Example 4 except that the membrane containing microorganism wasallowed to contain the mediator by the above two methods. The sensorswere applied for the measurement of BOD with the following method.

BOD standard liquid and a buffer solution (0.05 M phosphate buffersolution, pH 7.0) were so mixed as to prepare solutions with differentBOD concentrations. One of the BOD buffer solution of them was injectedinto the sample liquid accumulation portion of one of the microbialsensor prepared by the method (1), and the value of electric current wasmeasured. When this kind of measurement with the mediator containingmicroorganism membrane is used for the measurement of BOD, the mediatoris fixed on the electrode, and after a sample liquid is injected intothe sensor, the mediator will dissolve into the sample liquid.Therefore, once the sensor is used, it can not be used for succeedingmeasurements (or the mediator must be replenished for succeedingmeasurements). Then, a different sensor prepared, however, by the samemethod (1) was applied for the measurement of BOD of a BOD buffersolution having a different concentration from the foregoing. The sameoperation was repeated each time a fresh BOD buffer solution with adifferent concentration was subjected to measurement. Finally, all theBOD buffer solutions with different concentrations were measured oftheir values of electric current of BOD buffer solutions, and theresults are shown in FIG. 26.

In the same manner as in the above, the microbial sensors prepared bythe method (2) were applied for the measurement of values of theelectric current of BOD buffer solutions with different concentrations.The results are shown in FIG. 27.

From above results it is obvious that the use of the microbial electrodeincorporating a mediator gives the same results of the measurments asderived from a sample liquid supplemented with the mediator.

EXAMPLE 10 Measurement of the Concentration of Matters Contained inVarious Sample Liquids

Microbial electrodes were prepared in the same manner as in Example 1,to measure the concentration of the matters as listed in Table 1 withthe microorganisms as listed in Table 1.

                  TABLE 1                                                         ______________________________________                                        Matters                                                                       to be measured                                                                              Microorganisms                                                  ______________________________________                                        Glucose       Gluconobacter suboxydans IFO3172                                Glycerose     Pseudomonas fluorescens IFO14160                                Ethanol       Gluconobacter rubiginosus IFO3244                               L-glutamic acid                                                                             Pseudomonas fluorescens IFO14160                                Maltose       Pseudomonas pseudomallei ATCC15682                              Acetic acid   Bacillus subtilus IFO13719                                      Sucrose       Pseudomonas caryophylli IFO13591                                S. starch     Pseudomonas putida IFO14164                                     ______________________________________                                    

The cultivation of microorganisms were made as follows. The same methodwas employed as in Example 1 for the cultivation of Pseudomonasfluorescens IFO14160.

For the cultivation of Gluconobacter suboxydans IFO3172, Gluconobacterrubiginosus IFO3244 and Pseudomonas caryophylli IFO13591, theingredients as listed in Table 2 were mixed with purified water to 1 Lto prepare a culture medium (pH 7.0). To each of 100 ml of this medium,above microorganisms were inoculated respectively, and cultivated at 30°C. for 24 hours with shaking. The each culture was centrifuged toseparate the cells from the medium. The cells were washed with a buffersolution (0.05 M phosphate, pH 7.0), and suspended in the same buffersolution.

                  TABLE 2                                                         ______________________________________                                        Ingredient       Content (g)                                                  ______________________________________                                        Potato extract   200                                                          Condensed yeast  30                                                           Liver extract    25                                                           Meat extract      5                                                           Thioglycolate medium                                                                           10                                                           dehydrated                                                                    Glucose           5                                                           Glycerol         15                                                           Calcium carbonate                                                                              15                                                           ______________________________________                                    

For the cultivation of Pseudomonas putida IFO14164, the ingredients aslisted in Table 2 excluding calcium carbonate were mixed with purifiedwater to 1 L to prepare a culture medium (pH 7.0). To 100 ml of thismedium, the microorganism was inoculated, and cultivated at 30° C. for24 hours with shaking. The culture was centrifuged to separate the cellsfrom the medium. The cells were washed with a buffer solution (0.05Mphosphate, pH 7.0), and suspended in the same buffer solution.

For the cultivation of Bacillus subtilus IFO13719, the ingredients aslisted in Table 3 were mixed with purified water to 1 L to produce aculture medium (pH 7.0). To 100 ml of this medium, the microorganism wasinoculated, and cultivated at 30° C. for 24 hours with shaking. Theculture was centrifuged to separate the cells from the medium. The cellswere washed with a buffer solution (0.05 M phosphate, pH 7.0), andsuspended in the same buffer solution.

                  TABLE 3                                                         ______________________________________                                        Ingredient     Content (g)                                                    ______________________________________                                        Peptone        10                                                             Yeast extract   5                                                             Liver extract  25                                                             Glucose         3                                                             Glycerol       15                                                             Sodium chloride                                                                               3                                                             ______________________________________                                    

For the cultivation of Pseudomonas pseudomallei ATCC15682, theingredients as listed in Table 4 were mixed with purified water to 1 Lto prepare a culture medium (pH 7.0). To 100 ml of this medium, themicroorganism was inoculated, and cultivated at 37° C. for 24 hours withshaking. The culture was centrifuged to separate the cells from themedium. The cells were washed with a buffer solution (0.05 M phosphate,pH 7.0), and suspended in the same buffer solution.

                  TABLE 4                                                         ______________________________________                                        Ingredient     Content (g)                                                    ______________________________________                                        Beef extract    3                                                             Peptone        10                                                             Sodium chloride                                                                               5                                                             Glycerol       40                                                             ______________________________________                                    

Microbial electrodes were prepared in the same manner as in Example 1,by immobilizing the microorganisms cultivated as above on gold-depositedOmnimembranes with PVA-SbQ, to measure the concentration of matterscontained in various sample liquids below.

(1) Measurement of glucose concentration

The microbial electrode incorporating Gluconobacter suboxydans IFO3172cultivated as described above was applied to the same measurement systemas used in Example 6 to measure the concentration of glucose. First, acertain voltage was applied between the microbial electrode and thecounter electrode dipped in a sample liquid tank containing amediator-supplemented (20 mM potassium ferricyanide) buffer solution,and the value of electric current flowing therebetween was measured.Then, a standard liquid (a glucose solution comprising 100 mg ofglucose/10 ml) was added and the value of electric current was measuredin the same manner. The result was recorded with a recorder. The resultsare shown in FIG. 28.

(2) Measurement of glycerol concentration

The microbial electrode incorporating Pseudomonas fluorescens IFO14160cultivated as described above was applied to the same measurement systemas used in Example 6 to measure the concentration of glycerol. First, acertain voltage was applied between the microbial electrode and thecounter electrode dipped in a sample liquid tank containing amediator-supplemented (20 mM potassium ferricyanide) buffer solution,and the resulting electric current was measured. Then, a standard liquid(a glycerol solution comprising 100 mg of glycerol/10 ml) was added andelectric current was measured in the same manner. The result wasrecorded with a recorder. The results are shown in FIG. 29.

(3) Measurement of ethanol concentration

The microbial electrode incorporating Gluconobacter rubiginosus IFO3244cultivated as described above was applied to the same measurement systemas used in Example 6 to measure the concentration of ethanol. First, acertain voltage was applied between the microbial electrode and thecounter electrode dipped in a sample liquid tank containing amediator-supplemented (20 mM potassium ferricyanide) buffer solution,and the resulting electric current was measured. Then, a standard liquid(an ethanol solution comprising 100 mg of ethanol/10 ml) was added andelectric current was measured in the same manner. The result wasrecorded with a recorder. The results are shown in FIG. 30.

(4) L-glutamic acid

The microbial electrode incorporating Pseudomonas putida IFO14164cultivated as described above was applied to the same measurement systemas used in Example 6 to measure the concentration of L-glutamic acid.First, a certain voltage was applied between the microbial electrode andthe counter electrode dipped in a sample liquid tank containing amediator-supplemented (20 mM potassium ferricyanide) buffer solution,and the resulting electric current was measured. Then, a standard liquid(a L-glutamic acid solution comprising 100 mg of L-glutamic acid/10 ml)was added and electric current was measured in the same manner. Theresult was recorded with a recorder. The results are shown in FIG. 31.

(5) Maltose

The microbial electrode incorporating Pseudomonas pseudomallei ATCC15682cultivated as described above was applied to the same measurement systemas used in Example 6 to measure the concentration of maltose. First, acertain voltage was applied between the microbial electrode and thecounter electrode dipped in a sample liquid tank containing amediator-supplemented (20 mM potassium ferricyanide) buffer solution,and the value of electric current flowing therebetween was measured.Then, a standard liquid (a maltose solution comprising 100 mg ofmaltose/10 ml) was added and electric current was measured in the samemanner. The result was recorded with a recorder. The results are shownin FIG. 32.

(6) Acetic acid

The microbial electrode incorporating Bacillus subtilus IFO13719cultivated as described above was applied to the same measurement systemas used in Example 6 to measure the concentration of acetic acid. First,a certain voltage was applied between the microbial electrode and thecounter electrode dipped in a sample liquid tank containing amediator-supplemented (20 mM potassium ferricyanide) buffer solution,and the value of electric current flowing therebetween was measured.Then, a standard liquid (an acetic acid solution comprising 100 mg ofacetic acid/10 ml) was added and electric current was measured in thesame manner. The result was recorded with a recorder. The results areshown in FIG. 33.

(7) Sucrose

The microbial electrode incorporating Pseudomonas caryophylli IFO13591cultivated as described above was applied to the same measurement systemas used in Example 6 to measure the concentration of sucrose. First, acertain voltage was applied between the microbial electrode and thecounter electrode dipped in a sample liquid tank containing amediator-supplemented (20 mM potassium ferricyanide) buffer solution,and the value of electric current flowing therebetween was measured.Then, a standard liquid (a sucrose solution comprising 100 mg ofsucrose/10 ml) was added and electric current was measured in the samemanner. The result was recorded with a recorder. The results are shownin FIG. 34.

(8) S. starch

The microbial electrode incorporating Pseudomonas putida IFO14164cultivated as described above was applied to the same measurement systemas used in Example 6 to measure the concentration of S. starch. First acertain voltage was applied between the microbial electrode and thecounter electrode dipped in a sample liquid tank containing amediator-supplemented (20 mM potassium ferricyanide) buffer solution,and the value of electric current flowing therebetween was measured.Then, a standard liquid (a S. starch solution comprising 100 mg of S.starch/10 ml) was added and electric current was measured in the samemanner. The result was recorded with a recorder. The results are shownin FIG. 35.

From above results, it is obvious that according to the presentinvention it can be profitably to measure of the concentration ofvarious matters contained in various sample liquids, besides BOD.

Industrial Applicability

The microbial electrode and the microbial sensor of the presentinvention allow the measurement even in a solution poor in dissolvedoxygen, and enables direct measurement of the concentration of mattersin a solution without resorting to oxygen electrodes, thereby beingcapable of down sizing. Accordingly, the present invention is able toprovide a microbial sensor with reduced cost as production, which isdisposable and free from reduced precision in measurement due todeteriorated microorganisms after long use.

We claim:
 1. A microbial sensor for measuring a chemical or biologicalcomponent present in a solution, comprising:(a) a microbial electrodecomprising:an electric conductor; a microorganism cell layer fixed onand electronically contacted with the electric conductor to detecttransfer of electrons generated by the microorganism cells whenmetabolizing the chemical or biological component; and an insulatinglayer which insulates the electric conductor from the solution when inuse; (b) a counter electrode which is in contact with the solution whenused; (c) a support having two faces insulated from each other, whereinthe electric conductor of the microbial electrode is fixed on one of thefaces to sandwich said electric conductor between said microorganismcell layer and said support, and the counter electrode is fixed on theother face of said support; (d) a generator of electric potential whichgenerates an electric potential between the counter electrode and themicrobial electrode to facilitate transfer of electrons therebetweengenerated by the microorganism cells; and (e) a detector for measuringthe electric current passing between the counter electrode and themicrobial electrode; whereby the chemical or biological component ismeasured based on a predetermined correlation between the measuredelectric current and the amount of electrons transferred by themicroorganism cells when metabolizing the chemical or biologicalcomponent.
 2. The microbial sensor of claim 1, wherein saidmicroorganism cells are procaryotic cells selected from the groupconsisting of; E. coli, Bacillus, Gluconobacter, Pseudomonas, andActinomycetes.
 3. The microbial sensor of claim 1, wherein saidmicroorganisms are included into a gel membrane, photo-setting resinmembrane, or a polyacrylamide membrane, or a polymer membrane.
 4. Themicrobial sensor of claim 3, wherein said gel membrane is selected fromthe group consisting of; an alginate gel membrane, a carrageenan gelmembrane, an agarose gel membrane, a cudlan gel membrane and anachitosan gel membrane.
 5. The microbial sensor of claim 3, wherein saidphoto-setting resin membrane is a photo crosslinkable polyvinyl alcoholmembrane.
 6. The microbial sensor of claim 1, wherein said electricconductor is platinum, gold, silver, graphite, or carbon.
 7. Themicrobial sensor of claim 1 further comprising a mediator forfacilitating the transfer of electrons generating from the metabolism ofthe various chemical or biological components to the electrode.
 8. Themicrobial sensor of claim 7, wherein said mediator is selected from thegroup consisting of pigments, biological oxidation/reduction materials,and metal EDTA compounds.
 9. The microbial sensor of claim 8, whereinthe pigments are selected from the group consisting of1-methoxy-5-methylphenaxinium methylsulfonate, 2,6-dichloroindophenol,9-dimethylaminobenzo-a-phenazoxonium chloride, methylene blue,indigotrisulfonic acid, phenosafranin, thionine, new methylene blue,2,6-dichlorophenol, indophenol, azule B,N,N,N',N'-tetramethyl-p-phenylenediamine dihyrochloride, resorufine,safianine, sodium anthraquinone β-sulfonate, and indigo carmine.
 10. Themicrobial sensor of claim 8, wherein said biological oxidation/reductionmaterials are selected from the group consisting of riboflavin,L-ascorbic acid, flavin adenine dinucleotide, flavin mononucleotide,nicotine adenine dinucleotide, lumichrome, ubiquinone, hyroquinone,2,6-dichlorobenzoquinone, 2-methylbenzoquinone,2,5-dihyroxybenzoquinone, 2-hydroxy-1,4-naphtoquinone, glutathione,peroxidase, cytochrome C and ferredoxin.
 11. The microbial sensor ofclaim 8, wherein said metal EDTA compounds are selected from the groupconsisting of Fe-EDTA, MN-EDTA, Zn-EDTA.
 12. The microbial sensor ofclaim 7 wherein said mediator has a concentration of 40 nM to 100 mM.13. The microbial sensor of claim 12 wherein said mediator has aconcentration of 10 μM to 50 mM.
 14. The microbial sensor of claim 1further comprising a reference electrode used to set the potential ofthe microbial electrode.
 15. The microbial sensor of claim 1 furthercomprising additional microbial electrodes allowing for the synchronousmeasurement of a plurality of chemical or biological components.
 16. Themicrobial sensor according to claim 1, which is incorporated in a sampleliquid collecting unit comprising: a sample liquid-sucking inlet; aliquid accumulation portion for storing sample liquid sucked through thesample liquid-sucking inlet; and an aspiration pump portion forgenerating an aspirating power, wherein the microbial sensor is disposedin the sample liquid accumulation portion.